Second messengers and protein kinases play key roles in the signalling pathways that control the response of mammalian cells (and probably all eukaryotic cells) to most stimuli. Although such signalling pathways have been subjected to extensive studies, detailed knowledge on e.g. the exact timing and spatial characteristics of signalling events is often difficult to obtain due to lack of a convenient technology. There is, however, one exception to this rule: our understanding of the role of Ca.sup.2+ in e.g. intracellular signalling has been greatly improved due to the development of the fluorescent Ca.sup.2+ probe FURA-2 that permits real times studies of Ca.sup.2+ in single living cells.
Moreover, the construction of probes for cAMP (Adams et al., Nature 349 (1991), 694-697) and activity of the cAMP-dependent protein kinase (Sala-Newby and Campbell, FEBS 307(2) (1992), 241-244) has been attempted. The protein kinase A probe, however, suffers from the drawback that it is based on the firefly luciferase and accordingly produces too little light for fast single cell measurements. The cAMP probe on the other hand has to be microinjected and is therefore not well suited for routine laboratory work. In conclusion, both probes lack some of the elegant properties that resulted in the widespread use of FURA-2.
Recently it was discovered that Green Fluorescent Protein (GFP) expressed in many different cell types, including mammalian cells, became highly fluorescent (Chalfie et al., Science 263 (1994), 802-805). WO95/07463 describes a cell capable of expressing GFP and a method for selecting cells expressing a protein of interest and GFP based on detection of GFP-fluorescence in the cells.